Evidence for the Existence of Two ^-Carotene Pools and Two Biosynthetic ^-Carotene Pathways in the Chloroplast K. H. Grumbach Botanisches Institut der Universität Karlsruhe, Kaiserstraße 12, D-7500 Karlsruhe
Z. Naturforsch. 34 c, 1205— 1208 (1979); received September 4, 1979
Biosynthesis, Carotenoids, /?-Carotene, Photosynthesis Two /7-Carotene pools were obtained in the chloroplast. It is concluded that one pool is very small like a-carotene and responsible exclusively for the biosynthesis of /?-ionone-xanthophylls. The other ^-carotene pool is proposed to be the one, that is located close to photosystem I and is involved in photosynthesis as a light protecting agent for chlorophylls against photo-oxidation. Furthermore evidence is given that both /7-carotene pools are synthesized by independent biosyn thetic pathways.
Introduction chloroplast in two different pools which are proba bly synthesized by independent biosynthetic path The intraorganelle distribution of chlorophylls, ways. carotenoids and quinones in the chloroplast is differ ent. Chlorophylls are present exclusively in the thy lakoids [!]• Besides chlorophylls, carotenoids and Materials and Methods quinones are constituents of the photosynthetic Chlorella pyrenoidosa (211-81, algae collection membrane, but they are also present in the osmio- Göttingen) was grown autotrophically for 7—10 philic plastoglobuli [2, 3]. Furthermore investiga days and then supplied with [4,5-3H] leucine tions carried out by Douce reveal, that carotenoids (500 |iCi, specific activity l.OmCi/mmol) for a pe (violaxanthin) are contained even in the chloroplast riod of 18 h. During the final 40 min [2-14C] acetate envelope [4, 5]. (200 nCi, specific activity 55 mCi/mmol) was also Fractionation studies of chloroplast lamellae have supplied. Thereafter the cells were washed free from also obtained differences in the distribution of chlo exogenous labelled substrate and reincubated in the rophylls, carotenoids and quinones between photo light in fresh nutrition medium. At different times system I and II and the light-harvesting complex after the beginning of the experiment Chlorella cells [6 — 10]. Whereas /7-carotene and chlorophyll a are were harvested, carotenoids isolated, purified to a the major pigments of photosystem I-, chlorophyll a constant specific radioactivity and assayed for 14C- and lutein are present in large amounts in photosys and 3H-radioactivity by liquid scintillation counting. tem II-particles together with plastoquinone-9, a-to- Radish seedlings grown for 6 days in light were in copherol and lower concentrations of /7-carotene, cubated with [2-3H]mevalonate (1 mCi, specific activ violaxanthin and neoxanthin. In contrast to photosys ity 100 — 500 mCi/mmol) together with [2-14C] ace tem I and II every carotenoid present in the chloro tate (1 mCi, specific activity 40 — 60 mCi/mmol) for plast is contained in the light-harvesting complex to 24 h. Thereafter carotenoids were isolated, purified gether with chlorophyll a and b [10]. to a constant specific radioactivity and assayed for In this communication dual labelling experiments 14C- and 3H-radioactivity. with [3H]leucine/[14C] acetate and [3H] mevalonate/- [14C] acetate were achieved to obtain differences in the intraplastidic compartmentation of carotenoids Results in chloroplasts of green radish seedlings and Chlorel Besides leucine, C 02, acetate and mevalonate are la pyrenoidosa. The aim of these investigations was the most common precursors for carotenoid biosyn to indicate in vivo that /7-carotene is contained in the thesis in algae and higher plants. The first specific re action in terpenoid biosynthesis leading to the for Reprint requests to Dr. K. H. Grumbach. mation of carotenoids involves the biosynthesis of 0341-0382/79/1200-1205 $01.00/0 phytoene from geranylgeranylpyrophosphate [11]. 1206 K. H. Grumbach • Compartmentation of yS-Carotene in the Chloroplast
Phytoene Phytoene \ thesized directly from a-carotene can also be seen \ Phytofluene Phytofluene after the pulse chase when the 14C- and ^-radioactiv 1 i ♦ f ity is lost in a-carotene and at the same time increases 5-Carotene 5-Carotene I 1 in lutein. On the other hand zeaxanthin was labelled ♦ ♦ Neurosporene Neurosporene to a much higher extent by [3H] leucine and [14C] ace / \ / X tate than its precursor /7-carotene. During the last ot-Zeacarotene (3-Zeacarotene p -Z eac aro ten e Lyc 60 min of the experiment 14C-radioactivity decreases \ / I Lycopene 1 \ / in /7-carotene and increases in zeaxanthin due to the / \ transformation of [14C-/7] carotene into [14C] zeaxan >C arotene y-Carotene H-Carotene thin. 3H-radioactivity, however, was not lost to the i 1 ♦ » same extent but increased in /7-carotene and zeaxan ot-Carotene ß -C aro ten e i thin as well. The effect that the [14C]label was lost in * 1 Lutein Zeaxanthin /7-carotene to a greater extent than was ^-radioactiv I ity, as well as the higher mol specific 3H- and 14C-ra- Antheraxanthin dioactivity of zeaxanthin compared to /7-carotene is » Violaxanthin in contrast to the previous assumption that a carote » noid is contained in the chloroplast in one pool. It Neoxanthin can only be explained by the existence of two /7-caro- Fig. 1. General scheme for carotenoid biosynthesis [12] tene pools. From this it is concluded, that one /7-ca- and the proposed independent /?-carotene biosynthesis. rotene pool is present in the chloroplast as a small biosynthetic pool like a-carotene. This pool has a higher mol specific radioactivity than zeaxanthin, Phytoene then is desaturated via phytofluene, C-ca- whereas the other pool seems to be larger with a rotene and neurosporene to lycopene (Fig. 1). Lyco lower specific radioactivity. pene finally cyclizes to X- or y-carotene. On the other Further evidence that /?-carotene is contained in hand the cyclization of neurosporene to ß- and a-zea- the chloroplast in two different pools was obtained carotene and finally to X- or y-carotene was propos by dual labelling experiments with [2-3H] mevalona- ed as an alternative reaction. A-carotenc cyclizes to te and [2-14C] acetate in chloroplasts of green radish a-carotene, whereas y-carotene froms both a- and /?- seedlings. Besides this it was also investigated, wheth carotene. By introduction of oxygen a-carotene er these two /?-carotene pools are synthesized in one froms lutein, while /7-carotene is the precursor of zea or two independent biosynthetic pathways. It is xanthin. known that carotenoid biosynthesis proceeds via ge- It is generally believed that all carotenoids are ranylgeranylpyrophosphate and different acyclic ca synthesized in one biosynthetic pathway in the chlo rotene precursors like phytoene, phytofluene and ly roplast stroma and thereafter attached to tne thyla copene that cyclizes to X- and y-carotene and finally koid membrane. If a carotenoid is synthesized via to a- or /7-carotene. If carotenoids are synthesized in one biosynthetic pathway and is contained in the one biosynthetic pathway, then [14C] acetate and chloroplast in only one pool, then, in a pulse-chase- [3H]mevalonate are incorporated in the acyclic caro labelling experiment, the label in a carotenoid mole tenoid precursors and also in carotenes and xantho- cule that is derived from a terpenoid precursor like [3H] leucine should follow the same kinetic as that of another e. g. [14C] acetate resulting in a constant Table I. Mol-specific radioactivity of radish carotenoids after 24 hours incorporation with [2-14C] acetate and [2-3H]- 3H/14C-ratio. mevalonic acid as precursors. The 3H- and 14C-labelling kinetic of a- and /7-caro- Cafotenpidi {14qDPM/n«n©l 3h / 14c tSfie and their produett kite« anti äe£*aitä)ta fir«* Chlorella pyrenoidosa are shown in Fig. 2. After 18 h incubation time with [3H] leucine all carotenoids lycopene 208 x 10s 310X105 0.67 a-carotene 154X105 0 - were labelled by tritium. Mol specific radioactivity lutein 4x 10s 0 - was higher in a-carotene than in lutein reflecting their /7-carotene 24 x 105 0.9 X 10s 26.67 precursor-product relationship. That lutein is syn violaxanthin 6 x10s 0 - K. H. Grumbach • Compartmentation of /7-Carotene in the Chloroplast 1207
dpm /jmilS ol S 6 ß - CAROTENE yumol 101 10 ' >> > o * T t 1 n uc i 5 i h 10 § 20 40 60 90 120 180 min tight dpm dpm LUTEIN dpm yumol jumol j j f c j 6 ZEAXANTHIN jumol MO5 10 : -4P = Mo6 3H ______, 3h _____
______- o Lrfi =i
J 4i h 10 s 10 *T ---- r “ 1--- 1------1------1------r f- s 0 20 40 60 90 120 180 min 20 40 60 90 120 180 min light light Fig. 2. Labelling kinetic of a- and /7-carotene and their biosynthetic products lutein and zeaxanthin from Chlorella pyrenoi- dosa in a tracer kinetic experiment with [4,5-3H] leucine and [2-14C] acetate as precursors.
phylls after 24 h incubation time. As shown in Ta Discussion ble I, [3H] mevalonate and [14C] acetate were incorpo Chlorophylls and carotenoids are involved in pho rated in lycopene and ^-carotene whereas a-carote- tosynthesis as light absorbing agents. Besides chloro ne, lutein and violaxanthin were labeled only by phylls, /7-carotene and lutein are the major chloro [3H] mevalonate. The highest mol specific radioactiv plast pigments [12]. Whereas the function of chloro ity was obtained in lycopene and the lowest in lutein phylls as light harvesting pigments in the reaction and violaxanthin. This is consistent with the first ex centers of photosystem I and II and the light-harvest periment. ing complex is known [10], only little information is However, the differences obtained in the label of present concerning the function of carotenoids. lycopene and /7-carotene compared to a-carotene, lu Carotenoids are involved in the activity of photosys tein and violaxanthin in this experiment are not tem I and II [10, 12] but also in the xanthophyll cy conclusive with the fact that all carotenoids are syn cle [13]. Particularly /?-carotene is proposed to func thesized in the chloroplast in one biosynthetic path tion as a light protecting agent for chlorophylls way. They rather support the existence of two /7-ca- against photooxidation by deactivating chlorophyll rotene pools in the chloroplast and also the existence triplets or singlet oxygen [14]. Furthermore it was of two independent biosynthetic pathways for the pointed out, that a large ^-carotene pool is associ forMstio» of l-6araieae. There seems to exist a sat all ated with the ante**» pigmeats ©f pfeotesystcm I /7-carotene pol that is responsible exclusively for the [15]. biosynthesis of /7-ionone xanthophylls. Besides this The dual labelling experiments performed in this pool a larger /7-carotene pool is evident. This large communication, concerning the analysis of the intra- /?-carotene pool could be synthesized by an indepen plastidic distribution of carotenoids are in agree dent terpenoid pathway. ment with previous investigations and reveal that ß- 1208 K. H. Grumbach • Compartmentation of ^-Carotene in the Chloroplast carotene is contained in the chloroplast in different of incubation implies that this large /7-carotene pool pools. It is concluded, that one /?-carotene pool is as of photosystem I is synthesized by an independent small as a-carotene. As a biosynthetic pool this /2-ca terpenoid pathway (Fig. 2). For these investigations rotene could be used for the biosynthesis of ß-iono- represent only a preliminary result, further experi ne-xanthophylls. Besides this biosynthetic pool an ments concerning the intraplastidic distribution of other larger /7-carotene pool is present in the chloro carotenoids are in progress. plast. This large pool could be the /7-carotene pool that is located close to photosystem I and protects Acknowledgements the chlorophylls against photo-oxidation [14, 15]. Financial support by Royal Society London and The total discrimination of [14C] acetate compared to Deutsche Forschungsgemeinschaft is gratefully ac [3H] mevalonate in all radish xanthophylls after 24 h knowledged.
[1] J. T. O. Kirk and R. A. E. Tilney-Bassett, The Plastids, [8] A. Radunz and G. H. Schmid, Z. Naturforsch. 28 c, 2nd ed., pp. 64 - 97, Elsevier, Biomedical Press, Ox 36 - 44(1973). ford 1978. [9] L. H. Grimme, Ber. Deutsch. Bot. Ges. 87, 509 — 514 [2] H. K. Lichtenthaler, Protoplasma 68 ,65 - 77 (1969). (1974). [3] K. H. Grumbach and H. K. Lichtenthaler, Proc. 3rd [10] J. P. Thomber, Ann. Rev. Plant. Physiol. 26, 127 — 158 Int. Congr. on Photosynth. (M. Avron, ed.), Vol. I, pp. (1975). 515 — 523, Elsevier, Amsterdam 1975. [11] G. Britton, Chemistry and Biochemistry of Plant Pig [4] R. Douce, R. B. Holtz, and A. A. Benson, J. Biol. ments, 2nd (T. W. Goodwin, ed.), pp. 262 — 326, Aca Chem. 248,7215 - 7222 (1975). demic Press, London 1976. [5] R. Douce and J. Joyard, Advances in the Biochem. and [12] G. F. W. Searle and J. S. C. Wessels, Biochim. Bio Physiol, of Plant Lipids (L. A. Appelqvist and C. Lil- phys. Acta 504, 84 - 99 (1978). jenberg, eds.), pp. 79 — 89, Elsevier, Amsterdam 1978. [13] A. Hager, Ber. Deutsch. Bot. Ges. 88 ,27 - 44 (1975). [6] L. P. Vernon, B. Ke, H. Mollenhauser, and E. R. Shaw, [14] H. T. Witt, Quart. Rev. Biophys. 4,365 - 477 (1971). Progress in Photosynth. Res. (H. Metzner, ed.), Vol. I, [15] W. Junge, H. Schaffemicht, and N. Nelson, Biochim. pp. 137 - 148 IUBS, Tübingen 1969. Biophys. Acta 462,73 — 85 (1977). [7] H. K. Lichtenthaler and M. Tevini, Z. Pflanzenphys- iol. 6 2 ,3 3 - 5 0 (1970).